Hi guy, I am trying to use the DEqMS tool for my differential expression analysis. I followed the steps from the DEqMS-package-vignette until 2.1.4 - Make contrasts. This is the commands;

cond = as.factor(c(X1_Frozen,X2_Frozen,X3_Frozen,X4_Frozen,X11_RNAlater,X12_RNAlater,X13_RNAlater,X14_RNAlater)) #X1_Frozen etc. = equals row names/groups
design = model.matrix(~0+cond)
design = model.matrix(~0+cond)
colnames(design) = gsub("cond","",colnames(design))
contrast = makeContrasts(contrasts=x,levels=design)

When trying to run the contrast command I'll get the following error message:

Error in makeContrasts(contrasts = x, levels = design) : 
  The levels must by syntactically valid names in R, see help(make.names).  Non-valid names: -Inf,-4.00735369535212,-3.85423988827818,-3.57233660941963,-3.45430347451484,-3.44084465049062,-3.23991739765898,-3.13420477162754,-3.09740268774595,-3.06618643730488,-3.03005644431212,-3.01908649459226

etc. which seems like it can't handle negative value. However, I would expect negative values since it it log2 transformed and median centered, right? I appreciate any advice on how to potentially fix this. Thanks!

Cheers, Marlene

Hey Marlene,

The problem is likely created in your very first line:

cond = as.factor(c(X1_Frozen,X2_Frozen,X3_Frozen,X4_Frozen,X11_RNAlater,X12_RNAlater,X13_RNAlater,X14_RNAlater))

What are X1_Frozen, X2_Frozen, et cetera? - I presume that they are supposed to be column names?

Your cond vector should probably be something like this:

c('Frozen', 'Frozen', 'Frozen', 'Frozen', 'RNAlater', 'RNAlater', 'RNAlater', 'RNAlater')

, and this vector should align (in length and order) with the columns of your expression matrix.

Also note that you have duplicated this line: design = model.matrix(~0+cond)

Finally, is there a reason for including an intercept term (~ 0)?

Kevin

All you actually need is:

cond <- factor( rep(c("Frozen","RNAlater"), each=4))
design <- model.matrix(~cond)

There's no need for a contrast or for any editing of the column names.

You don't need to use makeContrasts at all, but I will mention that the error message you report lists what appears to be raw expression values, something that should not be entered to that function. It is hard to see how this particular error message could arise from the code you give.